Altered steroid milieu in AI resistant breast cancer ... · 7/9/2019 · 1 1 Altered steroid...
Transcript of Altered steroid milieu in AI resistant breast cancer ... · 7/9/2019 · 1 1 Altered steroid...
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Altered steroid milieu in AI resistant breast cancer facilitates AR mediated gene expression associated with poor 1
response to therapy. 2
Short title: Androstenedione drives AR mediated gene expression in AI resistance. 3
Laura Creevey1* ([email protected]) 4
Rachel Bleach1* ([email protected]) 5
Stephen F Madden2 ([email protected]) 6
Sinead Toomey3 ([email protected]) 7
Fiona T Bane1 ([email protected]) 8
Damir Varešlija 1 ([email protected]) 9
Arnold D Hill4 ([email protected]) 10
Leonie S Young1 ([email protected]) 11
‡Marie McIlroy1 ([email protected]) 12
1. Endocrine Oncology Research Group, Department of Surgery, RCSI, Dublin 2 13
2. Data Science Centre, RCSI, Dublin 2 14
3. Department of Oncology, RCSI, Beaumont Hospital, Dublin 9 15
4. Department of Surgery, RCSI, Beaumont Hospital, Dublin 9 16
* Both authors contributed equally to this manuscript 17
The authors declare there have been no competing interests. 18
‡Corresponding author: M. McIlroy ([email protected]) 19
Endocrine Oncology Research Group. 20
Department of Surgery, 21
Royal College of Surgeons in Ireland, 22
St. Stephens Green, 23
Dublin 2, 24
Ireland. 25
Tel No: 0035314022286 26
Funding: Health Research Board (HRA-POR-2013-276) (MMcI) and BHCRDT (MMcI). 27
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Abstract 28
Divergent roles for androgen receptor (AR) in breast cancer have been reported. Following aromatase inhibitor 29
(AI) treatment, the conversion of circulating androgens into estrogens can be diminished by >99%. We wished to 30
establish whether the steroid environment can dictate the role of AR and the implications of this for subsequent 31
therapy. 32
This study utilizes models of AI resistance to explore responsiveness to PI3K/mTOR and anti-AR therapy when cell 33
are exposed to unconverted weak androgens. Transcriptomic alterations driven by androstenedione (4AD) were 34
assessed by RNA-sequencing. AR and estrogen receptor (ER) recruitment to target gene promoters was evaluated 35
using ChIP and relevance to patient profiles was performed using publicly available datasets. 36
Whilst BEZ235 showed decreased viability across AI sensitive and resistant cell lines, anti-AR treatment elicited a 37
decrease in cell viability only in the AI resistant model. Serum and glucocorticoid-regulated kinase 3 (SGK3) and 38
cAMP-Dependent Protein Kinase Inhibitor β (PKIB), were confirmed to be regulated by 4AD and shown to be 39
mediated by AR; crucially re-exposure to estradiol suppressed expression of these genes. Meta-analysis of 40
transcript levels showed high expression of SGK3 and PKIB to be associated with poor response to endocrine 41
therapy (HR=2.551, p=0.003). Furthermore, this study found levels of SGK3 to be sustained in patients who do not 42
respond to AI therapy. 43
This study highlights the importance of the tumour steroid environment. SGK3 and PKIB are associated with poor 44
response to endocrine therapy and could have utility in tailoring therapeutic approaches. 45
Keywords 46
Androgen receptor, aromatase inhibitor, breast cancer, enzalutamide, SGK3. 47
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Introduction 56
ER positive tumours account for approximately 75% of all breast cancer diagnoses [1]. The emergence of selective 57
estrogen receptor modulators (SERM) in the 1970’s proved to be a rubicon in the fight against breast cancer; with 58
all first line therapies thereafter focusing on estrogen driven ER activity [2]. Since the mid -2000s AIs have been 59
recommended as first line therapy for hormone receptor positive, post-menopausal breast cancers [3]. However, 60
the development of resistance to these drugs is a perennial problem with disease recurrence in ~30% of patients 61
[4]. Mechanisms of resistance to anti-estrogen therapy are multifaceted and include alterations in co-activator 62
recruitment [5], dominance of growth factor pathways [6], upregulation of aromatase [7], ER mutations [8] and 63
alterations in steroid handling in breast tumour epithelial cells [9]. 64
The role of AR in breast cancer development and progression is somewhat mired in controversy with evidence 65
suggesting it can either antagonise or promote breast cancer depending on tumour context (reviewed[10]). In ER 66
positive breast cancer the general consensus is that AR protein is a positive prognostic indicator [11]. Conversely, 67
others have shown AR to take on the mantle of a pseudo ER, particularly in the setting of triple negative breast 68
cancer [12]. More recently, a potentiating role of AR in the development of endocrine resistance in ER positive 69
breast cancer has been emerging [13, 14]. Subsequently there is growing interest in targeting the AR with a 70
number of ongoing clinical trials assessing the utility of anti-AR drugs in the treatment of advanced breast cancer. 71
More recently, Aceto et al (2018) identified AR signalling to be activated in circulating tumour cells isolated from a 72
patient with breast to bone metastasis [15]. Nevertheless, when we consider that the majority of breast cancers 73
express AR protein, with some reports of >90% positivity [16], it makes understanding the dichotomous role of AR 74
all the more problematic. It is therefore imperative that we differentiate between AR functions which are 75
protective and those which are tumour promoting; whether this is dependent upon protein interactors, altered 76
steroid levels or mutations remains wholly unknown. 77
PI3K and mTOR signalling has been implicated in mechanisms of resistance to AI therapy in preclinical and clinical 78
trials [17]. In this context, AR in particular, has been established to play a prominent role in mediating PI3K and 79
mTOR signalling in a number of neoplasms [18]. AR mode of action in this setting is known to be quite disparate 80
from transcriptional steroid activity and may contribute to mechanisms of resistance to AI therapy. Indeed, non-81
genomic, sex-nonspecific actions of both estrogen and androgens have been demonstrated to activate intracellular 82
signalling pathways [19]. Here, we show that AR protein levels are elevated in cell line models of letrozole 83
resistance. Significantly, due to the mechanism of action of AI therapy, the intracellular environment becomes 84
saturated with androgens, and will become largely estrogen depleted. The risk that this alteration in the steroid 85
environment may facilitate resistance is supported by clinical evidence which has shown that serum levels of the 86
direct precursor steroid, androstenedione (4AD), are elevated in patients progressing on AI therapy [20, 87
21]. Understanding individual tumour intracrinology will be critical to evaluating this clinically as it is known that 88
elevated levels of androgens (4AD and DHEA) are associated with breast cancer risk >2 years prior to cancer 89
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detection. This suggests that hormone levels effect risk rather than hormone levels being altered by localized 90
steroid production [22]. 4AD is known to bind AR and can induce AR nuclear translocation in vitro, albeit with a 91
lower affinity than 5-Dihydrotestosterone (DHT) [23]. Herein, we report that this chronically altered androgenic 92
steroid environment enhances expression of SGK3 and PKIB transcripts which are both identified as AR/ER 93
regulated genes. SGK3 has previously been identified as a downstream target of both PI3K and AR in prostate 94
cancer [24] , highlighting it as a potential central mediator for both signalling pathways in AI resistant breast cancer 95
[25, 26]. This current study highlights the impact of steroid levels on transcriptional regulation and identified 96
SGK3, in particular as a potential indicator of poor response to AI therapy. As SGK proteins can be targeted 97
pharmacologically this pin-points SGK3 as a potential therapeutic target for ER positive breast cancer that may not 98
respond to conventional endocrine treatment. 99
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Materials and Methods 101
Cell culture. 102
Cell lines were cultured as follows: endocrine-sensitive MCF7 were grown in DMEM (low glucose) with 10% of 103
fetal bovine serum (FBS) and 100 U penicillin/0.1 mg ml−1 streptomycin (Pen-strep) plus 10−8 M 17-β-estradiol 104
(Sigma E8875). MCF7 derived AI-sensitive cells (MCF7-Aro) were developed in-house and cultured in phenol red 105
free MEM (Sigma Aldrich, UK), 10% charcoal dextran stripped FCS, 1% Pen-Strep, 1% L-Glutamine and 200 µg/ml 106
G418 (Geneticin). MCF7-Aro derived letrozole-resistant cells (MCF7-Aro-LetR) were created by long-term 107
treatment of MCF7-Aro with letrozole and 4AD >3 months (Novartis, Basel, Switzerland) in charcoal dextran 108
stripped FCS, 1% Pen-Strep, 1% L-Glutamine, 200 µg/ml G418, 2.5-8 M 4AD and 10-6 M letrozole [27]). An obvious 109
morphologic change was noted in the transition of MCF7 cells to AI resistant MCF7aro-LetR with cells displaying a 110
large increase in cell surface area. To further assess the impact of the steroid environment on these cells they were 111
then maintained in medium supplemented with estradiol (10-8 M for 15 weeks) and designated as MCF7aro-LetR-112
Est. After 15 weeks, MCF7aro-LetR-Est cells had reverted to smaller cell sizes with cobblestone morphology, as is 113
observed in MCF7 cells grown in estradiol. 114
ZR75.1 were included as an additional luminal A model that express high AR. They are known to have a PTEN 115
mutation and were cultured in MEM, 10% FCS, 1% Pen-Strep, 1% L-Glutamine [28]. ZR75.1-derived AI-sensitive 116
cells (ZR-Aro) were developed in-house by lentiviral transduction of ZR75.1. ZR-Aro-derived letrozole resistant cells 117
(ZR-Aro-LetR) were then generated by long term treatment of ZR-Aro with 4AD and letrozole >3months using the 118
same culture medium as above and are fully characterized (supplemental figure 2 A, B & C). All cells were 119
maintained in steroid depleted medium for 72 hours before treatment with steroids or drugs. All cells were 120
incubated at 37°C under 5% CO2 in a humidified incubator. In-house cells were authenticated and are routinely 121
verified as endocrine resistant, mycoplasma free and all cells are utilised within 10 passages for triplicate 122
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experiments. The Aro-cell lines from which the AI-resistant cells are derived are generally not used as comparators 123
in these studies as it is well established that CYP19 amplification is a hallmark of endocrine resistance [29] and if 124
cultured in the absence of AI and steroid these cells are even less endocrine sensitive [30-32]. 125
Western blotting 126
Cells were harvested, lysed, electrophoresed, and immunoblotted with specific primary antibodies and 127
corresponding horseradish peroxidase-conjugated secondary antibody (Dako, Den). See supplemental materials 128
and methods for antibody detail. 129
MTS assay 130
For drug treatments, MCF7 and MCF7-Aro-LetR cells were steroid-depleted for 72 hours. MCF7 cells were plated in 131
steroid depleted media supplemented with estradiol 10-8 M to enable cell growth and MCF7-Aro-LetR were seeded 132
in steroid depleted media prior to the addition of BEZ235 (Selleck Chemicals) as per optimal dose concentration, 133
enzalutamide (10µM) (Selleck chemicals) or DMSO vehicle (Sigma Aldrich). Treatments were replenished every 3 134
days. Colorimetric outputs were analyzed by measuring the absorbance at 490nm using a spectrophotometer 135
(Perkin Elmer, USA). Prior to transfection MCF7-Aro-LetR and ZR-Aro-LetR cells were steroid-depleted for 72 136
hours. Cells were transfected with siRNA against SGK3 (L-004162-00, Dharmacon, Colorado, USA) or siRNA 137
negative control (d-001206-13-05) and seeded in a 96-well plate prior to the addition of 4AD (10-7 M). Two-way 138
ANOVA with Bonferroni post-test was used for statistical analysis of results for treatment 1- 3 day. Student’s t-test 139
(two-tailed) was used to compare means for siRNA experiments. 140
Colony forming assay 141
Assays were performed as per previously described [14]. 142
RNA extraction, library preparation and RNA-sequencing 143
To assess the transcriptional effects of 4AD in endocrine resistant breast cancer cells, RNA-sequencing was 144
performed using MCF7-Aro-LetR cells which were steroid depleted for 72 hours and treated with either 4AD (10-7 145
M) or vehicle in the presence of letrozole 10-6 M in triplicate. RNA was extracted using an RNeasy Kit (Qiagen, 146
Hilden, Germany). Sequencing was performed on an Illumina HiSeq technology (minimum 10 million clean reads 147
of the RNA-quantification/sample). Three independent biological libraries were prepared for each sample to 148
facilitate the expression detection and variance estimation. 149
Transcriptomic analysis (RNA-seq analysis and microarray analysis) 150
RNA seq pre-processing: see supplemental material and methods. 151
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Microarray pre-processing: see supplemental material and methods. 152
For both transcriptomic experiments differential expression was determined using the ebayes function of the R 153
package Limma [33]. In each case a binary comparison was performed between 4AD treated cells and controls. In 154
the case of the microarray experiment the following samples were included, GSM1016474, GSM1016475 and 155
GSM1016476 (controls) and GSM1016486, GSM1016487 and GSM1016488 (treated). A p-value of less than 0.05 156
and a fold change greater than 1.4 fold was considered significant. The package Limma was chosen here for 157
differential expression analysis as it is particularly robust when dealing with small sample sizes in both microarray 158
and RNA-seq experiments [34]. These two lists of differentially regulated genes were overlapped with each other 159
and data from a publically available list of genes associated with acquired endocrine therapy resistance in breast 160
tumours expressing ESR1 but not ERBB2 [35]. The original authors referred to this as their “group 4 set” of genes 161
which exhibited strong hormone responsiveness. A detailed description of how it was generated can be found in 162
the original manuscript. This data was used to generate a candidate genes list for further investigation. All 163
calculations were carried out in the R statistical environment (https://cran.r-project.org/). 164
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RNA-seq validation 166
A panel of breast cancer cell lines (MCF7, MCF7-Aro-LetR, MCF7-Aro-LetR-Est, ZR75.1 and ZRaro-LetR) were steroid 167
depleted for 72 hrs followed by treatment with 4AD (10-7 M) and ethanol vehicle (0.0001% (v/v)) for 24hr. 168
Letrozole 10-6 M was also added to AI resistant cells lines. RNA was extracted using an RNeasy Kit (Qiagen, Hilden, 169
Germany). cDNA synthesis was performed using Superscript III First Strand Synthesis System (Life Sciences). See 170
supplemental material and methods for primer details. 171
MassArray analysis 172
See supplemental material and methods. 173
siRNA transfection 174
AR, ESR1 and SGK3 were silenced by transient transfection using experimentally verified pools of siRNA. AR was 175
silenced using siRNA pools SMARTpool (catalog number L-003400-00) (30 nM), SGK3 SMARTpool (catalog number 176
L-004162-00) (30 nM) and ESR1 SMARTpool (catalog number L-003401-00-0005) (30 nM), all purchased from 177
Dharmacon. All transfections were carried out using Lipofectamine 2000 transfection reagent according to 178
manufacturer’s instructions (Invitrogen, UK) and a non-targeting siRNA pool negative control (Dharmacon) was 179
used as a control for all siRNA experiments. Confirmation of mRNA knockdown was performed using primers for 180
AR (F: 5’-AGCGACTTCACCGCACCTCA-3’, R: 5’-CAGTCTCCAAACGCATGTCCCCG-3’), ESR1 (F: 5’ 181
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TGTACCTGGACAGCAGCAAG-3’, R: 5’-TCTCCAGGTAGTAGGGCACC-3’) 24 hours post transfection. siRNA efficacy 182
was also confirmed at protein level (Supplemental Figure 2D&E). 183
Chromatin immunoprecipitation. 184
MCF7-Aro-LetR cells were treated with 4AD 10-7 M (30 mins), estradiol 10-8 M (45 mins) or ethanol vehicle. ChIP 185
was performed as previously described [27]. Rabbit anti-AR (3 ug, sc-816, SCBT), anti-ESR1 (6 ug, SC-543, SCBT) or 186
mouse IgG (6 ug)/rabbit (3 ug) (Dako) was added to the supernatant fraction and incubated overnight at 4°C with 187
rotation. Proteins were un-crosslinked, DNA extracted and specific primers used to amplify the DNA of the SGK3 188
proximal promoter. SGK3 proximal promoter primers: Forward 5’-GACTTGTGTAACATGGTCTCTTTCA-3’ and reverse 189
5’-CAAGTTCAATCTGACCCTCATATCT-3’ [24]. 190
Meta-analysis of SGK3 mRNA expression and breast cancer patient survival. 191
BreastMark [36] is an algorithm that enables the identification of subsets of gene transcript/miRNAs that are 192
associated with disease progression and survival in breast cancer and its subtypes. Using this resource the 193
association of expression levels of SGK3 mRNA and survival were evaluated in endocrine-treated/untreated 194
datasets. 195
Evaluation of SGK3 in clinical cohorts and responsiveness to endocrine therapy. 196
Series GSE59515 (Accurate Prediction and Validation of Response to Endocrine Therapy in Breast Cancer)[37] was 197
used to assess mRNA expression levels of SGK3 and AR pre and 3 months post AI treatment in a cohort of post-198
menopausal breast cancer patients (n=25). USCS Xena browser (https://xenabrowser.net/heatmap/) was used to 199
interrogate TCGA Breast cancer datasets filtered to focus analysis on pre and post-menopausal, endocrine therapy 200
treated patients (n=415). Copy number variation for SGK3 was evaluated and cohorts were stratified by masked 201
copy number repeat to eliminate sex chromosome and germ-line artifacts. Kaplan Meier graphs (quartiles) were 202
generated from these data to ascertain association of copy number amplification and overall survival. 203
RNA extraction from FFPE tissues 204
Breast cancer patients (n=6) who were responsive (n=3) or non-responsive (n=3) to AI therapy were selected. 205
Informed written consent was received from all patients, and the study was approved by institutional review board 206
Royal College of Surgeons in Ireland (CTI09/07). Hematoxylin and eosin stained sections of formalin fixed, paraffin 207
embedded (FFPE) tumor tissues were analyzed by a pathologist for histological and tumor cellularity classifications. 208
Tumor content was annotated on sections and RNA was extracted from these areas using Qiagen AllPrep DNA/RNA 209
FFPE kit according to manufacturer instructions. 210
Statistical analysis 211
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A two-tailed Student’s t test was used to compare means. Two-way ANOVA with Bonferroni post-test was used to 212
compare replicate means. Dose response curves were normalized to 100% and 0% viability based on the lowest 213
and highest drug concentrations (GraphPad Prism v8). 214
Data accession code 215
RNA-seq data are available at the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/), provisional 216
accession code: SRP148035. 217
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Results 219
AI-sensitive MCF7 and AI-resistant MCF7Aro-LetR cells exhibit decreased cell viability when treated with BEZ235, 220
however only MCF7-Aro-LetR cells display decreased cell viability when treated with anti-AR therapy. Estradiol 221
dependent parental MCF7 cells were found to harbor a PIK3CA E545K missense mutation (Figure 1A and B (i)) 222
which is a known hotspot mutation in breast cancer [38]. Analysis of the letrozole resistant MCF7 derivative cell-223
line (MCF7-Aro-LetR) confirmed they retained the same mutation (Figure 1A and B (ii)). The cell lines exhibited 224
varied levels of AR protein expression with low amounts in the MCF7 and higher levels in the MCF7-Aro-LetR 225
(Figure 1C and supplemental Figure 1A (i)). This was mirrored in the ZR75.1 letrozole resistant derivative cell line 226
(ZR75aro-LetR) when compared to their parental cell line; ER protein levels did not vary across the panel of cell 227
lines (Figure 1C and supplemental Figure 1A (ii)). Dose response analysis was used to establish sensitivity to 228
PI3K/mTOR inhibition by the pan class inhibitor BEZ235 (Sellekchem) and to a αδ PI3K inhibitor, Pictilisib 229
(Sellekchem) (Supplemental Figure 1B (i-iv) & C (i-ii)). 230
Endocrine sensitive (MCF7) and AI resistant cells (MCF7-Aro-LetR) were treated with BEZ235 or enzalutamide (anti-231
AR) as single or combination treatment followed by an evaluation of cell viability. BEZ235 treatment significantly 232
decreased cell viability in all cell lines tested compared to vehicle (Figure 1D and 1E). Of note, the AI-resistant 233
MCF7-Aro-LetR cells showed a significant decrease in levels of cell viability following enzalutamide treatment 234
(Figure 1E), this was in contrast to the parental MCF7 cells, in which viability was sustained when AR was targeted 235
compared to vehicle (Figure 1D). Combined BEZ235 and enzalutamide treatment resulted in a significant decrease 236
in viability across all cell lines, however, this effect was most likely carried by BEZ235 (Figure 1D and 1E). This 237
result was mirrored by colony forming assays suggesting that it is cell proliferation that is being affected (Figure 1F 238
(i-ii)). 239
4AD upregulates genes associated with steroid and PI3K signalling. 240
Transcriptomic analysis enabled the identification of genes differentially regulated when AI resistant cells are 241
exposed to 4AD in the presence of letrozole to recapitulate the steroid environment under AI therapy 242
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(Supplemental table 1, RNAseq Data). This in-house generated gene list was further supported by comparison with 243
genes identified in a publically available dataset of MCF7-Aro cell treated with 4AD without AI. This helped define 244
gene expression changes attributed solely to the effects of 4AD and provided evidence of putative 4AD specific 245
regulation in AI-resistance. Subsequent comparison with the ‘Group 4’ genes associated with ER+, HER2- endocrine 246
resistance (GSE8140, [35]) yielded a subset of 8 genes (Figure 2A). As using data from independently published 247
datasets introduces confounding factors the genes identified were validated in-house in two genetically disparate 248
AI resistant models. 249
Chronic adaptation to the steroid environment induces a unique transcriptome that can be reverted by re-250
introduction of estradiol. 251
Validation of the transcriptomic analysis was performed by treating endocrine sensitive MCF7 and ZR75.1 cells and 252
their endocrine resistant derivatives (MCF7-Aro-LetR and ZR-Aro-LetR cells) with 4AD and investigating the impact 253
on the transcript levels of the top 4 genes in our list: growth regulation by estrogen in breast cancer 1 (GREB1), 254
serum /glucocorticoid regulated kinase family member 3 (SGK3), cAMP-dependant kinase inhibitor β (PKIB) and 255
MYB proto-oncogene like 1 (MYBL1). Each of these genes validated in both AI-resistant cell lines, MCF7-Aro-LetR 256
and ZR-Aro-LetR, with significant increases in GREB1, SGK3, PKIB and MYBL1 in response to 4AD treatment (Figure 257
2B (i-viii)). GREB1, SGK3, PKIB and MYBL1 did not increase in response to 4AD treatment in endocrine sensitive 258
MCF7 cells and no change was observed for 3 out of 4 of these genes in endocrine sensitive ZR75.1 cells with the 259
exception of PKIB which demonstrated a very modest increase (Figure 2C (i-viii)). Overexpression of the aromatase 260
gene was used to generate both models of AI resistance and has also been a feature observed in the development 261
of AI resistance in the clinic. In order to address whether CYP19A overexpression was a driving factor in the 262
increased expression of SGK3, PKIB, MYBL1 and GREB1 a series of experiments using MCF7 cells overexpressing 263
aromatase and cultured in the presence of 4AD were performed. Data from these experiments confirmed that 264
aromatase overexpression alone did not induce increased levels of these genes (Supplemental figure 3A (i-iv)). 265
Further evidence that aromatase overexpression is inconsequential for these genes is provided via analysis of GSE 266
10911 [39] and from TCGA cohorts (Supplemental figure 3B and supplemental figure 6). The former study focused 267
on MCF7-Aro cells cultured in the absence of steroid, with testosterone (T) or with T plus AI. SGK3 levels were 268
increased in the MCF7aro plus T compared to steroid unstimulated cells, most interestingly, there was a further 269
significant increase in SGK3 mRNA in cells cultured with T and AI. Reversion of the MCF7-Aro-LetR transcriptional 270
response to 4AD could be achieved by culturing MCF7-Aro-LetR in estradiol for 15 weeks. Cell growth initially 271
decreased with notable alteration in cell morphology to the cobblestone appearance of estradiol dependent MCF7 272
and smaller cell size (see Figure 6A for illustrative images). Treatment of these cells with 4AD (24 hours) produced 273
very minimal effects on gene expression in comparison to those observed in the MCF7-Aro-LetR from which they 274
were derived (Figure 3A (i-iv), 3B & 6A). 275
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Androstenedione stimulated induction of target gene mRNA is reduced post siRNA knockdown of either AR or 276
ER; both AR and ER are recruited to a common region on each gene promoter. 277
As it has been established that many transcripts are under cooperative AR:ER regulation [13] we then wished to 278
establish whether the genes identified in this current study were regulated by ER and/or AR. To determine if AR 279
and/or ER are integral to the 4AD mediated upregulation of GREB1, SGK3, PKIB and MYBL1, siRNA inhibition of AR 280
and ER was performed, and siRNA efficacy was confirmed (supplemental Figure 2D (i-ii) & 2E (i-ii)). The impact of 281
knockdown of nuclear receptors (NRs) on target gene expression in MCF7-Aro-LetR cells was evaluated. siRNA 282
knockdown of AR combined with 4AD treatment resulted in a significant decrease in SGK3 and PKIB transcript 283
levels but had no significant impact on GREB1 or MYBL1 (Figure 4A (i-iv)). siRNA knockdown of ER combined with 284
4AD treatment resulted in significantly reduced levels of all four targets (Figure 4B (i-iv)). Regulation of SGK3 285
protein by AR and ER was then confirmed at the level of protein expression (supplemental figure 3C (i-ii)). AR and 286
ER recruitment to these target genes was then evaluated in a publically available dataset (GSE104399) [40]. 287
Although primarily a male breast cancer dataset it contains ChIP-seq data for both AR and ER recruitment in a 288
female breast cancer patient (patient 8: ER+, PR+, HER2-, post-menopausal) (Figure 4C, upper panels). Using the 289
ChIP-seq peak information published by the original authors we were able to confirm the binding of ER to the 290
proximal promoter of SGK3, GREB1, MYBL and PKIB and AR to GREB1 and PKIB in this patient sample (see 291
supplemental table 2 and supplemental figure 4 for additional detail for binding locations). Enhanced AR 292
recruitment and ER occupancy of the SGK3 promoter was confirmed using ChIP in 4AD stimulated MCF7-Aro-LetR 293
(Figure 4C and 4D). Whilst this analysis was limited to one patient, it does suggest that AR and ER bind at regions 294
enriched with ESR1 motifs. ER ChIP-seq data for the MCF7-Aro-LetR confirmed ER recruitment to the proximal 295
promoters of all 4 genes in the presence of 4AD plus letrozole (Figure 4C, lower panel; supplemental table 2 and 296
supplemental figure 4). 297
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Knockdown of SGK3 inhibits AI resistant cell viability in the presence of androstenedione or estradiol. 299
SGK3 has previously been documented as a downstream target of AR and ER (α and β isoforms) in prostate and 300
ERα in breast cancer [24, 25] and is also associated with stabilization of endoplasmic reticulum stress. Here, we 301
have shown that SGK3 mRNA and protein levels are significantly increased in MCF7-Aro-LetR cultured in the 302
presence of 4AD (Figure 5A (i-ii)). Treatment with BEZ235 or a combination of the anti-AR therapy, bicalutamide, 303
with BEZ235 decreased SGK3 mRNA expression (Figure 5B). We found that when SGK3 levels are abrogated by 304
siRNA targeting SGK3 (Figure 5C (i-iii)) in the absence of steroid there was no change in cell viability (Figure 5D (i)), 305
in contrast, when SGK3 was targeted for degradation in the presence of 4AD there was a significant decrease in cell 306
viability (Figure 5D (ii)). This result was also reflected in cell counts of MCF7-Aro-LetR cells exposed to siRNA 307
targeting SGK3 (Figure 5D (iii)). Further validation was carried out in ZR-Aro-LetR cells (Supplemental figure 5A (i-308
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iii)) Follow-up siSGK3 experiments using a variety of steroids demonstrated that decreased viability is only evident 309
when cells are exposed to 4AD or estradiol but there is no alteration observed when cells are cultured in the 310
potent androgen, R1881, or the control, cholesterol (Supplemental figure 5B). 311
SGK3 and PKIB are associated with poor recurrence free survival (RFS) in the post-menopausal, endocrine 312
treated breast cancer patient population (ER+ PR+). 313
In vitro data presented has shown the impact of the steroid microenvironment upon target gene expression 314
identified by RNA-seq (schematic overview: Figure 6A). In post-menopausal women 100% of sex hormones are 315
synthesized in peripheral tissues from circulating adrenal and ovarian precursors steroids. Clinical data has shown 316
levels of 4AD to be elevated in patients whose disease progresses on AI therapy [19, 20] and more recent data has 317
shown 4AD dominates breast tumour intracrinology [41]. In contrast serum and tissue levels of estradiol are 318
markedly reduced in patients treated with an aromatase inhibitor [3]. Our study identified SGK3 and PKIB as 4AD 319
regulated transcripts mediated in part by AR in collaboration with ER. This supports the hypothesis that 320
transcriptional reprogramming as a result of the steroid environment can facilitate resistance to AI therapy 321
(summarized: Figure 6B). The BreastMark meta-analysis resource was used to evaluate the impact of SGK3 and 322
PKIB transcript levels on endocrine treated breast cancer patient outcome [36]. The high expression group in each 323
plot (blue) accounts for the upper quartile of expression levels for a particular transcript and the low expression 324
group (red) the remaining 75%. This was applied to each of the individual datasets and the information was then 325
combined to perform a global pooled survival analysis. Meta-analysis of the AR mediated genes, SGK3 and PKIB, 326
showed that these genes have no impact on RFS in treatment naïve, postmenopausal ER positive breast cancer, 327
however, when we evaluate their impact in an endocrine treated population it is clear that patients with high 328
levels of these transcripts do not benefit from endocrine therapy (Figure 6C (i-ii)). 329
SGK3 levels are sustained in breast cancer patients who do not respond to AI therapy. 330
To evaluate these observations further, SGK3 levels were assessed in a clinical cohort of breast cancer patients 331
defined as responsive or non-responsive to AI therapy. Data Series GSE59515 [37] was used to assess mRNA 332
expression levels of SGK3 and AR pre and 3 months post AI treatment in a cohort of post-menopausal breast 333
cancer patients (n=25). SGK3 levels are lower in responsive patients post therapy (drop in mean expression: 16.75 334
0.563, one-tailed t-test: p=0.0015 ) (Figure 7A(i)). Conversely, SGK3 mRNA in non-responders is somewhat 335
sustained (drop in mean expression 16.64 6.44, one-tailed t-test: not significant) (Figure 7A(ii)). This was 336
mirrored by levels of AR mRNA with a significant drop in responders (one-tailed t-test: p=0.0196) compared with 337
sustained levels in non-responders to AI therapy (one-tailed t-test: not significant) (Figure 7B (i-ii)). These data 338
were validated in a second cohort of AI responders and non-responders (n=6). SGK3 mRNA was only detectable in 339
post-menopausal non-responders (Figure 7A(iii), a result that is mirrored by the levels of AR mRNA (Figure 7B(iii). 340
Of note, the only patient non-responsive to AI with no detectable SGK3 or AR transcript was pre-menopausal; 341
highlighting the post-menopausal hormone state as being crucial to the expression of these target genes. Whilst 342
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exploring the expression of SGK3 transcript in clinical cohorts, it was noted that there is a large percentage (16%) 343
of breast cancer patients with an alteration in the SGK3 gene. The impact of SGK3 copy number amplification was 344
then evaluated in post-menopausal, breast cancer patients treated with endocrine therapy (n=132). Kaplan Meier 345
survival curves showed that copy number amplification of SGK3 significantly associates with poor survival in the 346
post-menopausal patients (p=0.016). Analysis of the pre-menopausal patient cohort yielded no association, 347
however, it should be noted that numbers in this cohort were <50 (Figure 7C). 348
349
Discussion 350
PI3K signalling as a driver of endocrine resistance in ER positive breast cancer has been validated in many previous 351
studies and clinical trials [17, 42]. Given that the combination of the αδ-PI3Kinase inhibitor pictilisib plus 352
fulvestrant does not improve survival in AI resistant breast cancer, suggests that the PI3K pathway alone or in 353
combination with ER is not the sole driver of AI resistance [43]. Our cell line models of AI resistant breast cancer 354
are cultured in letrozole and 4AD which results in elevation of AR protein levels in contrast to parental cell lines. In 355
light of the abundance of data from the field of prostate cancer which has elucidated mechanisms by which AR can 356
act as a mediator of second messenger signalling, a potentiating role for AR in our model of AI resistance was 357
evaluated [44]. In this study we have utilised transcriptomic analysis to elucidate gene expression changes 358
associated with resistance to AI therapy. 359
AI therapy creates a unique androgenic environment that permits exploration of steroid drivers of resistance that 360
may not be wholly dependent upon a functional ER; this is exemplified clinically by the similar overall survival 361
achieved when AI or selective estrogen receptor degrader (SERD) are used as second line therapy after recurrence 362
on AI [45]. AR is normally associated with good patient outcome [11, 16] and the pro-survival response observed 363
in the enzalutamide treated MCF7 cells in this study would fully support this role. However, the strongly opposing 364
action of the same drug in the isogenic MCF7-Aro-LetR cells highlights the importance of the steroid 365
microenvironment in directing the action of AR. As we have observed contrasting impact on cell growth with anti-366
AR therapy we may presume the response is heavily influenced by the steroid driver (estradiol in MCF7 and 367
androstenedione in MCF7-Aro-LetR). Our study is not without limitations as the cell line models used are 368
engineered to overexpress CYP19 (amplification of which is known to be associated with AI resistance [29]); 369
however, control experiments clearly show that aromatase overexpression in this system does not increase 370
expression of the 4AD target genes. Furthermore, analyses of clinical TCGA datasets show no association of CYP19 371
amplification and SGK3 expression, suggesting that this is a disparate mechanism of resistance. It is therefore 372
plausible that estrogen depletion as a consequence of AI therapy may be permissive of AR mediated 373
transcriptomic alterations. 374
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Elevated levels of sex steroids and their prohormones have long been associated with increased breast cancer risk 375
primarily via epidemiological and clinical observation [20, 46]. In this study we have focused on the effects of 4AD 376
arising from the adrenal gland and ovarial interstitial cells which acts as the direct sex-steroid precursor for both 377
androgens and estrogens [47, 48]. Disappointingly , clinical trials of the CYP17 inhibitor, AA (abiraterone), alone or 378
in combination with AI have shown no difference in progression free survival between modalities; results that are 379
perhaps confounded by an associated rise in progesterone and the potential of AA to act as an ER agonist in breast 380
cancer [49, 50]. It is also plausible that 4AD will be metabolized to other steroids such as 5α-androstanedione or 381
3β-androstanediol, themselves known to act as drivers of breast cancer proliferation [51]. Interestingly, a 382
proliferative role of androstene-3b,17b-diol has recently been suggested for ER positive breast cancer with low 383
intratumoural estradiol levels [52]. Whilst weaker sex steroids are incapable of inducing the conformational 384
change required for classical NR genomic action, they and/or their metabolites may play a role in driving non-385
genomic activity. Indeed, the sheer overabundance of weaker steroids and the formation of transient nuclear 386
receptor (NR) associations may well be able to initiate second messenger signalling which is not dependent upon 387
low disassociation constants [19]. It has often been reported that male and female sex steroids are associated 388
with both ER positive and ER negative breast cancers, highlighting the importance of the steroid drivers which may 389
be acting independently of the classical ER action in some tumours [46]. 390
As the majority of breast cancers express AR it is of interest to understand how the estradiol depleted steroid 391
environment, which accompanies AI therapy, modifies AR action. Many studies have focused on the 392
transcriptomic role of AR in this setting with much of the data indicating a co-operative AR:ER dynamic [13, 40]. 393
Indeed from the RNA sequencing data in our current study the main network of genes altered in response to 4AD 394
treatment were also found to be transcriptionally regulated by ER alpha. Of note, SGK3 and PKIB were also 395
confirmed to be regulated, in part, by AR. Recruitment of both NRs to regions harboring classical EREs in the 396
proximal promoter region was further confirmed in ChIP sequencing data from a female breast cancer patient in 397
the Severson study [40]. Importantly we have established that SGK3 is indeed a steroid regulated, AR target gene 398
and that its expression is enhanced when estrogen synthesis is inhibited in models of AI resistance. 399
The most interesting observation made in this study was the shift in gene expression induced by 4AD which was 400
shown, not only in AI resistant cells derived from MCF7, but also those generated from ZR75.1. This was in stark 401
contrast to the unresponsive parental cell lines, suggesting that chronic alterations in steroid bioavailability can 402
induce transcriptomic reprogramming. This was further supported by data from the long-term estradiol treated 403
MCF7-Aro-LetR, whose gene expression response to 4AD was completely reverted to reflect that of estradiol 404
dependent MCF7. SGK3, in particular, has recently been associated with poor response to AI therapy in breast 405
cancer and has been demonstrated to play a role in stabilization of the endoplasmic reticulum during cell stress 406
[25]. SGK3 has a high degree of similarity to AKT with both kinases targeting many of the same substrates. The SGK 407
family has been implicated in breast cancer resistance to PI3K inhibition in numerous in vitro studies and in a 408
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14
clinical trial [26]; of note SGK3 can be activated independently of Class I PI3K [53]. In our study, reduction in SGK3 409
transcript resulted in a loss of cell viability but only in the presence of 4AD or estradiol. This mirrors the finding 410
reported by Wang et al (2017), wherein they observed complete loss in viability when SGK3 is depleted; the more 411
pronounced effect reported was likely due to the increased efficacy of knockdown achieved and also because of 412
the different steroid environments (testosterone versus 4AD) [25]. 413
To understand the clinical relevance of these findings, a meta-analysis of the AR mediated genes, SGK3 and PKIB, 414
was performed. This analysis showed that these genes have no impact on RFS in treatment naïve, 415
postmenopausal, ER positive breast cancer, however, when we evaluate their impact in an endocrine treated 416
population it is clear that patients with high levels of these transcripts do not benefit from endocrine therapy. 417
When this was evaluated in AI responsive versus non-responsive patients it was clear that levels of SGK3 and AR 418
mRNA are sustained in patients failing on therapy. It was also noted that many breast cancer patients have 419
elevation of SGK3 copy number; whether this is as a consequence of an altered steroid microenvironment is an 420
intriguing possibility that is yet to be determined. We would purport, from these observations, that AI treated 421
breast cancers adapt to utilize bioavailable steroids such as those of adrenal origin. As highlighted in this study, 422
evaluating the implications of steroid alterations in the clinical management of disease is warranted. 423
424
Acknowledgements: Author contributions: LC carried out RNA-seq sample preparation, EC50 assays, MTS viability 425
assays, ChIP, qRT-PCR, participated in study design, analysed results and assisted in manuscript preparation. RB 426
assisted in validation of RNA-seq, MTS assays, western blotting, analysed results and generated graphics and 427
assisted in manuscript preparation. SM carried out bioinformatic analysis, assisted in the study design and 428
manuscript preparation. ST carried out MassArray analysis of cell line PI3K mutational status, assisted with study 429
design and preparation of manuscript. DV generated the ZR-Aro-LetR cell line, conducted the related motility and 430
PS2 expression analysis and assisted with preparation of manuscript. ADKH identified appropriate clinical samples, 431
prepared samples for analysis and assisted with study design. LY contributed to data analysis and manuscript 432
preparation. MMcI conceived study design, performed siRNA experiments, data analysis, generated graphics and 433
wrote the manuscript. All authors read and approved the final version of the manuscript. Many thanks to Dr 434
Katherine Sheehan, Tony O’Grady and Joanna Fay (Department of Pathology), Lance Hudson and Aisling Hegarty 435
for their guidance and help with sample preparation. Funding: Health Research Board (HRA-POR-2013-276) 436
(MMcI) and BHCRDT (MMcI). 437
Availability of data: The datasets generated and/or analyzed during the current study are available in the Gene 438
Expression Omnibus repository, [Accession code: SRP148035]. 439
Ethics approval and informed written consent to participate/ consent for publication – CTI 09/07 440
441
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15
442
443
Abbreviations 444
androgen receptor (AR) 445
aromatase inhibitor (AI) 446
estrogen receptor (ER) 447
androstenedione (4AD) 448
dehydroepiandrosterone (DHEA) 449
5-Dihydrotestosterone (DHT) 450
tissue microarray (TMA) 451
serum and glucocorticoid-regulated kinase 3 (SGK3) 452
cAMP-Dependent Protein Kinase Inhibitor β (PKIB) 453
selective estrogen receptor modulators (SERM) 454
growth regulation by estrogen in breast cancer 1 (GREB1) 455
MYB proto-oncogene like 1 (MYBL1) 456
nuclear receptors (NRs) 457
chromatin immunoprecipitation (ChIP) 458
formalin fixed paraffin embedded (FFPE) 459
recurrence free survival (RFS) 460
progression free survival (PFS) 461
abiraterone (AA) 462
testosterone (T) 463
464
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465
466
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53. Bago R, Malik N, Munson MJ, Prescott AR, Davies P, Sommer E, Shpiro N, Ward R, Cross D, 633 Ganley IG et al: Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the 634 phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class 635 III phosphoinositide 3-kinase. Biochem J 2014, 463(3):413-427. 636
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Figure legends. 646
Figure 1: AI-sensitive MCF7 and AI-resistant MCF7-Aro-LetR cells exhibit decreased cell viability when treated 647
with BEZ235, however only MCF7-Aro-LetR cells display decreased cell viability when treated with anti-AR 648
therapy. A. Tabulated details on two sets of paired isogenic breast cancer cells lines (endocrine sensitive MCF7, 649
ZR75.1 and their AI-resistant derivatives MCF7-Aro-LetR and ZR-Aro-LetR) including luminal type, AR protein 650
expression and mutations impacting PI3K signalling. B. (i) Traces from the Sequenom MassArray show that 651
endocrine sensitive MCF7 cells have a heterozygous PIK3CA E545K mutation and (ii) Endocrine resistant MCF7-Aro-652
LetR cells have a heterozygous PIK3CA E545K mutation. C. Western blot analysis was used to evaluate AR and ER 653
expression across five breast cancer cell lines (MCF7, MCF7-Aro-LetR, ZR75.1 and ZR-Aro-LetR) D & E. The effect of 654
pan class PI3K/mTOR inhibitor (BEZ235) and anti-AR (enzalutamide) on cell viability was assessed using MTS and 655
colony formation assay in endocrine sensitive and endocrine resistant cells. D MTS of MCF7 response to BEZ235 or 656
to a combination of BEZ235 plus enzalutamide compared to vehicle control. F (i) Colony formation assays reflect 657
MTS results in MCF7 cells. E MTS of MCF7-Aro-LetR cells in response to single agents BEZ235 and enzalutamide and 658
also to a combination treatment. F (ii) Colony formation assays reflect MTS results in MCF7-Aro-LetR cells. Graphs 659
representative of n=3. Error bars are representative of mean ± standard error of the mean (SEM). Two-way 660
ANOVA with Bonferroni post-test to compare replicate means was used in MTS day 1 to day 9 readings to 661
determine significance. Student’s paired, 2-tailed t-test established significance to compare means * p<0.05, ** 662
p<0.01, *** p<0.001. 663
Figure 2. 4AD upregulates genes associated with steroid and PI3K signalling in two models of AI resistance but 664
not in the parental cell lines. A. RNA sequencing was performed following androstenedione (4AD 10-7M) 665
treatment plus letrozole for 24 hrs in AI resistant MCF7-Aro-LetR cells versus vehicle. This data was then 666
compared with array data for MCF7aro treated with 4AD versus vehicle to identify 4AD specific transcripts. These 667
genes were then integrated with the ‘Group 4 ‘ gene set of endocrine resistant genes associated with ER+, HER2 –668
ve disease yielding a list of 8 genes. B. (i-viii) qRT-PCR validated differentially expressed genes (GREB1, SGK3, PKIB 669
and MYBL1) in AI resistant cell models MCF7-Aro-LetR and ZR-Aro-LetR cells. C. (i-viii) qRT-PCR showed expression 670
of these genes in parental MCF7 and ZR75.1 cells on exposure to 4AD . Graphs representative of n=3. Error bars 671
are representative of mean ± standard error of the mean (SEM). Student’s paired, 2-tailed t-test established 672
significance * p<0.05, ** p<0.01, *** p<0.001. ¶ [32], $ [36]. 673
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Figure 3. Culturing AI resistant cells (MCF7-Aro-LetR) in estradiol for 15 weeks reverts 4AD driven gene 674
expression. A. (i) & (ii) qRT-PCR of SGK3 mRNA displays a very modest increase, whereas GREB1 is significantly 675
decreased compared to vehicle control. A (iii) & (iv) PKIB and MYBL1 expression is unchanged in the presence of 676
4AD. Graphs representative of n=3. Error bars are representative of mean ± standard error of the mean (SEM). B. 677
Gene expression (SGK3, GREB1, PKIB & MYBL1) after 24 hrs 10-7M 4AD treatment in MCF7-Aro-LetR cells long 678
term cultured in 4AD compared to MCF7-Aro-LetR cells long term cultured in 10-8M estradiol (MCF7aroLetR-Est). 679
Student’s paired, 2-tailed t-test was carried out to compared individual gene expression across both cell lines. 680
Figure 4: Androstenedione stimulated transcript levels of SGK3 and PKIB are reduced in response to siRNA 681
knockdown of either AR or ESR1; in addition, AR and/or ER are recruited to these target genes in vivo and in 682
vitro. A. qRT-PCR of MCF7-Aro-LetR transfected with siRNA-AR treated with 4AD shows a significant decrease in 683
SGK3 and PKIB transcript levels (i-ii) but does not impact transcript levels of GREB1 or MYBL1 (iii-iv). B. qRT-PCR of 684
MCF7-Aro-LetR transfected with siRNA–ESR1 in MCF7-Aro-LetR cells treated with 4AD results in a significant 685
decrease in transcript levels of the SGK3, GREB1, PKIB and MYBL1 (i-iv). C. Recruitment of AR and ER to target 686
gene promoters was evaluated using publically available data from a breast cancer patient ChIP-sequencing study, 687
and ER recruitment in the MCF7-Aro-LetR cell line via ChIP-sequencing. ARϕ recruitment to the SGK3 and MYBL1 688
promoter was validated via ChIP in MCF7-Aro-LetR D. (i) Validation of the putative AR target gene SGK3 was 689
confirmed by performing ChIP to determine recruitment of AR to the promoter of SGK3 in MCF7-Aro-LetR cells in 690
the presence of 4AD. (ii) ChIP experiments were performed to determine recruitment of ER to the promoter of 691
SGK3 in MCF7-Aro-LetR cells treated with 4AD. All graphs representative of three experimental replicates. Error 692
bars are representative of mean ± standard error of the mean (SEM) of n=3. Student’s paired, 2-tailed t-test 693
established significance * p<0.05, ** p<0.01, *** p<0.001. 694
Figure 5: BEZ235 or combination of BEZ235 with the anti-AR Bicalutamide decreases SGK3 mRNA expression. 695
Knockdown of SGK3 inhibits AI resistant cell proliferation only in the presence of androstenedione. A (i) SGK3 696
protein expression is increased in the presence of 10-7M 4AD in MCF7-Aro-LetR cells. (ii) Densitometry analysis of 3 697
independent western blots confirmed significant increase in SGK3 protein expression with 4AD treatment. B. qRT-698
PCR evaluation of SGK3 mRNA expression with BEZ235 +/- anti-AR therapy bicalutamide in MCF7-Aro-LetR cells. C 699
(i) Western blot of SGK3 protein levels following siSGK3in MCF7-Aro-LetR. (iI) Densitometry analysis shows SGK3 700
protein levels following siSGK3. (iii) qRT-PCR of SGK3 mRNA following siSGK3 in MCF7aro-LetR D (i) MTS of 701
MCF7Aro-LetR cell viability following knockdown of SGK3 in the absence of steroid. (ii) MTS assay of MCF7aro-LetR 702
cell viability following siSGK3 combined with 4AD treatment . (iii) Cell counts confirmed decreased cell viability in 703
MCF7Aro-LetR cells following siSGK3. Graphs representative of n=3. Error bars are representative of mean ± 704
standard error of the mean (SEM). Student’s 2-tailed t-test established significance for A, B and D. 705
Figure 6: SGK3, PKIB and GREB1 are 4AD regulated transcripts mediated in part by AR in collaboration with ER. 706
Associated outcome in clinical cohorts highlights a significant impact on therapeutic response to endocrine 707
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therapy. A Schematic overview of key study findings highlight the morphologic and transcriptional changes driven 708
by chronic exposure of MCF7 to 4AD in the presence of letrozole when stably overexpressing CYP19. These same 709
cells, when maintained in estradiol, revert to their original morphology and no longer exhibit alterations in 4AD 710
mediated gene expression. B. Study summary: 1. In post-menopausal women 100% of sex hormones are 711
synthesized in peripheral tissues from circulating adrenal and ovarian precursors steroids. Clinical data has shown 712
levels of 4AD to be elevated in patients whose disease progresses on AI therapy and more recent data has shown 713
4AD dominates breast tumour intracrinology. 2. In contrast serum and tissue levels of estradiol are markedly 714
reduced in patients treated with an aromatase inhibitor. 3. Our study identified SGK3 and PKIB as 4AD regulated 715
transcripts mediated in part by AR in collaboration with ER. C (i) Meta-analysis of 4AD regulated genes SGK3 and 716
PKIB showed that there is no impact on recurrence-free survival in the endocrine untreated population (ER+ PR+, 717
n=379, HR= 1.23 (0.76 - 2.08), logrank p=0.37). (ii) Meta-analysis of 4AD regulated genes SGK3 and PKIB showed 718
that SGK3 and PKIB is associated with poor recurrence-free survival in the endocrine treated population (ER+ PR+, 719
n=231, HR=2.55 (1.34-4.85), logrank p=0.003). 720
721
Figure 7: Validation of SGK3 expression in clinical cohorts as an indicator of poor response to AI therapy. 722
A (i) Evaluation of GSE59515 shows SGK3 mRNA decreases significantly in patients who are responsive to AI 723
therapy (mean expression: 16.75 0.563, one-tailed t-test: p=0.0015). A (ii) Conversely, SGK3 mRNA in non-724
responders is somewhat sustained (mean expression 16.64 6.44, one-tailed t-test:not significant). A (iii) 725
Validation was performed in a second cohort of patients who were either responsive or non-responsive to AI 726
therapy. SGK3 mRNA was only detectable in non-responders. B (i) This was mirrored by levels of AR mRNA with a 727
significant drop in responders (mean 43.7822.07 one-tailed t-test: p=0.0196) compared with sustained levels in 728
B (ii) non-responders to AI therapy (mean 22.8729.98, one-tailed t-test: not significant). B (iii) Validation was 729
performed in a second cohort of patients who were either responsive or non-responsive to AI therapy. AR mRNA 730
was only detectable in non-responders. C. UCSC Xena browser was used to interrogate TCGA breast cancer data. 731
Kaplan Meier survival curves showed that copy number amplification of SGK3 significantly associates with poor 732
survival in endocrine treated, post-menopausal patients (p=0.016). Analysis of the pre-menopausal patient cohort 733
yielded no association, however, it should be noted that numbers in this cohort were <50. 734
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Published OnlineFirst July 9, 2019.Mol Cancer Ther Laura Creevey, Rachel Bleach, Stephen F Madden, et al. therapy.mediated gene expression associated with poor response to Altered steroid milieu in AI resistant breast cancer facilitates AR
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